Disinfecting teat care compositions

ABSTRACT

The present invention relates to novel compositions which are used to produce nitrous acid, in preferred aspects such compositions comprise a protic acid and a metal nitrite, and to methods for using these compositions, in particular for disinfecting mammalian teat skin.

RELATED APPLICATIONS

This application claims priority from U.S. application Ser. No.10/780,435, filed Feb. 17, 2004 and provisional application U.S.60/511,916, filed Oct. 17, 2003, which are incorporated by reference intheir entirety herein.

FIELD OF THE INVENTION

The present invention relates to novel compositions which are used toproduce nitrous acid and to methods for using these composition, inparticular for disinfecting mammalian teat skin.

BACKGROUND OF THE INVENTION

One of the single, most powerful controls that a dairy farmer can availhimself of, is routine pre- and post-milking teat dipping. Thisprocedure can dramatically reduce the incidence of mastitis in his dairyherd. Mastitis is by far the most prevalent and costly disease affectingdairy herds. More than half of the dairy animal population is thought tobe affected by bovine mastitis to some degree. Mastitis causes alowering of milk output and a reduced milk quality, accounting forlosses in the U.S. alone approaching $2 billion, a major portion ofwhich results from the lowered milk output of infected cows.

Mastitis is literally an inflammation of the mammary gland principallycaused by invasion of bacteria through the teat orifice at or aroundmilking times. During the milking process, it results from transfer tothe teat, from cow-to-cow or cow-to-human-to-cow, of so-calledContagious microorganisms from contaminated equipment and hands; or atother times, when the teat orifice remains open post-milking, as aresult of contact with so-called Environmental microorganisms thatdeposit on the teat and udder between milking periods. The Contagiousorganisms are primarily two, Staphylococcus aureus and Streptococcusagalactiae, while Environmental organisms include Streptococcus uberis,Streptococcus dysgalactiae, Klebsiella pneumoniae and Escherichia coli.The latter are present in the cow's surroundings, including the soil,its bedding, feces, and contaminated water.

Treatment of mastitis is more costly, and often ineffective, andgenerally involves antibiotic therapy. During such periods and for anumber of days thereafter, the cow's milk must be discarded. Dairymenuniformly agree that washing and/or disinfecting teats before milking,and dipping the teats afterwards, can markedly reduce the transfer ofenvironmental organisms to the teat, by self-infection, or bypost-milking with teat dips to control the transfer of contagiousorganisms from the equipment and hands. The teat dips (a term usedherein to include teat sprays as well) generally embody antimicrobialagents which are capable of at least substantially reducing in number,all such pathogens, and these dips additionally contain such optionalagents as humectants, thickening and/or barrier-forming agents, andcolorants. Without such disinfection, the individual teat quarters aresubject to a greater probability of becoming mastitic, causing problemsranging from lower milk quality and poorer milk yields to the actualdeath of the afflicted animal.

Teat dips, which are almost invariably liquid compositions, are mostoften single solutions or suspensions, used directly by the dairyman bywithdrawal of a day's portion from a larger container. Two-part teatdips have also been employed since 1987, where the active antimicrobialagent is formed by combination of certain chemical compounds in bothparts shortly before application. The antimicrobial agents most oftenfound in single part systems include iodophors, quaternary ammoniumcompounds, organic sulfonates, and chlorhexidine. The dual-part teatdips generally contain a chlorous acid system

Chlorous Acid Teat Dips: These two part teat dips are based on thegeneration of chlorous acid and/or chlorine dioxide by combination of ametal chlorite in one part and an acid source in the other, to formchlorous acid. The subsequent degradation of this acid into a series oftransient, cidal oxidants provides a heretofore unparalleled means forkilling or inactivating a broad spectrum of bacteria, yeasts, molds andviruses in a very rapid manner, and in high numbers. It is generallyaccepted that the acidified chlorite system is the most up-to-date andeffective antimicrobial in teat dip compositions.

More specifically, with regard to the metastable chlorous acid system,the chlorous acid molecule, HClO₂, represents a relatively smallfraction of the total chlorite ion present, typically no more than about15%. This tends to minimize the otherwise rapid degradation of thesystem. The antimicrobially-effective chlorous acid systems function atpH values from about 3.5 down to about 2.6. The protic acid source toeffect this conversion is generally an organic acid (U.S. Pat. Nos.4,986,990, 5,185,161), although inorganic acids (U.S. Pat. No. RE36,064) and even acid-inducing metal salts have been taught (U.S. Pat.No. 5,820,822), in the extended series of patents which disclose thevarious aspects of this technology. The acidified chlorite compositionswere first taught by Alliger in 1978 (U.S. Pat. No. 4,084,747) and in1982 (U.S. Pat. No. 4,330,531), where the acid activator was lacticacid, which was deemed critical to the unique activity of theacid/chlorite system. Subsequent prior art taught the creation of adiverse range of acidified chlorite compositions and their method ofuse. These included U.S. Pat. No. 4,891,216 (for topical application);U.S. Pat. No. 4,956,184 (for genital herpes); U.S. Pat. No. 5,100,652(for oral hygiene); U.S. Pat. No. 5,384,134 (for anti-inflammatoryactivity); U.S. Pat. Nos. 5,389,390 and 6,063,425 (for disinfectingpoultry and other meats); U.S. Pat. Nos. 5,597,561 and5,651,977(adherent topical disinfectants); U.S. Pat. No. 5,628,959(sterilizing hemodialyzers); U.S. Pat. No. 5,772,985 (bovine warts);U.S. Pat. No. 6,096,350 (for honey bee diseases); and U.S. Pat. No.6,123,966 (stabilized disinfecting compositions).

Chlorous acid: Positives and Negatives: One of the present inventors,Robert Kross, has worked extensively for a number of years in this areaof technology (he is a named inventor on the above-described patents),and as such, he has become very familiar with the capabilities anddeficiencies of the acid/oxy anion system, based upon chlorite. Althoughthe capabilities of the acidified chlorite system are extensive, severalinherent characteristics are present which limit its application incertain situations. The major difficulty lies in the relatively strongoxidizing tendency of the system, and in particular the corrosiveeffects of the chlorine dioxide (ClO₂), which forms upon degradation ofthe chlorous acid. ClO₂ will corrode many of the metals used in dairyspray equipment as well as those used in fabrication of medical anddental equipment, and those used to dispense the food disinfectingsolutions. A further detriment of the acidified chlorite systems is thenoxiousness of the ClO₂ gas, for which OSHA has listed a very lowpermissible concentration in the air to which workers may be exposed foran 8 hour period. That level, 0.1 parts per million in the air, is 10times lower, for example, than for chlorine, for which OSHA has listed amaximum permissible level of 1.0 ppm over an 8-hour period.

Alternative Two-Part Oxyanion System: In researching the seeminguniqueness of the uninegative chlorite ion, it became evident that thereis another oxyanion, namely the nitrite ion, that is similar to that ofchlorite (see, for example, Friedman's “On the Ultraviolet AbsorptionSpectra of Uninegative Ions,” re: the electronic properties of bothions). Both form unstable acid counterparts, i.e. chlorous and nitrousacids, with increasing instability as the acid form represents a growingfraction of the acidified oxyanion solution. Neither acid can beisolated. Nitrogen appears in at least 8 oxidation states in its watersoluble species; chlorine has at least 6. In general, species such asnitrous and chlorous acids, which have intermediate oxidation numbers,will be unstable with respect to disproportionation. The degradation ofboth of these acids leads to the formation of gases (chlorine dioxideand nitric oxide [NO]) which are unique in possessing unpairedelectrons. Both of these materials have unusual properties.

Chlorine dioxide has become an excellent replacement for chlorine inwater disinfection, by virtue of its high biocidal activity withoutformation of chloro-organic mutagens. It has also found use in thedisinfection of food. In both these cases the chlorine dioxide degradesthrough several steps, through a 5-electron transfer, to innocuouschloride ion. With respect to nitric oxide, while it is one of thesimplest biological molecules in nature, it has recently found its wayinto nearly every phase of biology and medicine. This ranges from itsrole as a critical endogenous regulator of blood flow and thrombosis, toa principal neurotransmitter mediating erectile function, to a majorpathophysiological mediator of inflammation and host defense. Thesemajor discoveries have stimulated intense and extensive research into avast array of fields including chemistry, molecular biology, and genetherapy.

One difference between the two paramagnetic, unpaired-electron moleculesof chlorine dioxide and nitric oxide, which derive respectively from thedegradation of chlorous acid and nitrous acid solutions, is that thechlorine in chlorine dioxide has lost one electron with respect to thatin chlorous acid (i.e. a +4 charge in the former vs. +3 in the latter),whereas the nitrogen in nitric oxide, with a +2 charge, has gained anelectron as cf. the +3 charge of the nitrite nitrogen. This differencemay lead to an advantage in the potential metal corrosivity of nitrousacid/nitric oxide systems versus chlorous acid/chlorine dioxide systems.In particular, a nitrous acid-based teat spray may well be lesscorrosive on metal spray equipment than a chlorous acid teat spray.

It appeared appropriate therefore, by virtue of the similar chemistry ofthe acidified nitrite as cf. the chlorite system, and its possible rolein producing antimicrobial activity paralleling that of acidifiedchlorite, to investigate, determine, and optimize the acidified nitritesystem as a disinfecting agent for use in teat dips. The specific intentwould be to reduce or eliminate those microorganisms which may otherwisecontaminate the teats of cows and other milk-producing species, frommilking equipment, hands, and the environment, that could otherwise leadto infection of the mammary gland (i.e. mastitis).

Although nitrous acid compositions for the treatment of infection havebeen proposed in the art, their applicability for use as teat dipcompositions has not been proposed. The prior art composition requirethat the two components are admixed at the intended environment of use(i.e. the diseased tissue) to release the NO and NO₂ which are thepurported active agents. If nitrous acid compositions are to be used forteat dips, on the other hand, it would be necessary to premix thecomponents up to one day prior to use, and have the resulting mixturecontinue to function antimicrobially. Stability of such solutions wouldoptimally allow for their use for at least a week, so that dairy farmerswould not have to continually discard excess teat dip mixtures that havenot been applied to the animals, and thus prepare fresh mixtures atleast once per day. If the farmer could “top off” remaining mixtureswith fresh components, it would save considerably in both time and cost.

To that end, to determine the functionality and applicability ofacidified nitrite systems for teat dip or spray use, the followingprogram was instituted. Germicidal activity was evaluated as a functionof the nitrous acid/nitrite ratios in test solutions, as controlled byavailable hydrogen ion. Success in this endeavor would be achieved if:

a)—suitable acid:nitrite combinations were found that were appropriatelygermicidal against the contagious and environmental organisms associatedwith mastitis;

b)—both nitrite and acid phases would accommodate, and be sufficientlycompatible with, other agents generally used in teat dip formulations;and

c)—most importantly, the resulting acidified nitrite systems had thelongevity of action following mixture that it could be used for at leastone day thereafter, while maintaining sufficient high germicidalactivity that it would be economically viable.

Accordingly, the present invention resulted from a search for acontrollable acidified nitrite antimicrobial system to parallel or evenexceed the superior qualities of the acidified chlorite system, as hasbeen manifest in the number of successful teat dips based thereon. If,indeed, some of the negative qualities of acidified chlorite teat dipscould be improved upon, such as the tendency to lose color intensity,generate noxious odors, corrode metal parts, and lose significantactivity within a few hours, the search would be deemed that much moresuccessful.

OBJECTS OF THE INVENTION

It is, therefore, an object of the present invention to provideantimicrobial acidified nitrite solutions for use as disinfectants inexternal teat care products.

It is a further object of the invention to control the antimicrobialaction of these solutions by modifying the nitrite concentration andacidity, and thus the degree of conversion of nitrite ion to nitrousacid, the presumptive source of germicidal action.

It is a further object of the invention to increase the length of timeover which the antimicrobial action of these solutions is exerted, bymodifying the nitrite concentration and acidity of these solutions.

It is yet a further object of the invention to provide compositionsbased upon nitrous acid which exhibit rapid bacterial kill and a broadspectrum of action against representative species of the variousmicrobial types which are of particularly concern in udder health.

It is still another object of the invention to provide acidified nitritesolutions which exhibit significant antimicrobial activity and to reduceand/or completely avoid corrosion of equipment associated with themilking process, including spray devices, milking claws, storage tanks,and pipe lines in comparison to chlorous acid systems.

It is an additional object of the invention to provide acidified nitritesolutions which are well tolerated by animal tissues, particularly thoseof teat skin.

These, and/or other objects of the present invention will becomeapparent from a review of the following summary of the invention anddescription of the preferred embodiments.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition fordisinfecting animal teats and milking-associated equipment, using anitrous acid generating composition. This composition comprises anaqueous solution containing a suitable amount of a protic acid and asuitable amount of a metal nitrite. The nitrite ion concentration (molepercent) in the form of nitrous acid is no more than about 95% by weightof the total amount of nitrite ion concentration. In certain preferredaspects of the present invention, the concentration (mole percent) ofnitrous acid is no less than about 10% and no greater than about 67%(about two-thirds) of the total amount of nitrite ion concentration.

In a preferred embodiment of this aspect of the present invention, thereis provided a composition for disinfecting the teats of the mammaryglands and milking-associated equipment with a composition comprising anitrous acid generating compound with a sufficient amount of a suitableorganic acid to lower the pH of the composition to less than about 4.5.The preferred organic acid is an α-hydroxy acid having a pKa rangingfrom about 2.8 to about 4.8 which preferably has the formula:

Where R¹ and R² may be the same or different and may be selected fromthe group consisting of hydrogen, methyl, —CH₂COOH, —CH₂COO—, —CH₂OH,—CHOHCOOH, —C₆H₅, and —CH₂C₆H₅.

In another preferred embodiment of this aspect of the present invention,there is provided a composition for disinfecting the teats of mammaryglands and milking-associated equipment with a composition comprising anitrous acid generating compound with an amount of phosphoric acid(pK_(a)=2.15) sufficient to lower the pH of the composition to less thanabout 4.5.

In another preferred embodiment of this aspect of the present invention,there is provided a storage stable nitrous acid composition fordisinfecting the teats of mammary glands and milking-associatedequipment with a composition comprising a nitrous acid generatingcompound, where the composition maintains adequate germicidal activityfor a period of at least one week and preferably at least about threeweeks after its preparation.

In another aspect, the present invention provides processes fordisinfecting the teats of mammary glands and milking-associatedequipment using the compositions described above. These processescomprise applying the compositions described above to the teats ofmammary glands and milking-associated equipment by dipping, spraying, orimmersion as appropriate, in order to disinfect the substrate.

In yet another aspect, the present invention provides a process forpreparing these disinfecting compositions and separately, fordisinfecting the teats of mammary glands and milking-associatedequipment using the resulting nitrous acid containing composition. Theprocess comprises contacting the protic acid with the metal nitrite toform the disinfecting compositions, which are used in effective amountsto disinfect the desired surface.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In describing the present invention, the following terms will be used.

The term “nitrite” or “nitrite salt” is used throughout thespecification to describe a salt of nitrous acid which is readilysoluble in an aqueous system and which readily dissociates into nitriteanion and counterion (generally, metal). Two particularly preferredsalts of nitrites for use in the present invention include sodiumnitrite and potassium nitrite, although a number of other nitrite saltsmay also be used in the present invention. The term “nitrite” is usedthroughout the specification to describe the form in which an amount ofa water soluble salt of nitrous acid either in dry or liquid state(preferably, as an aqueous solution) is added to the acid. In general,the nitrite is added to the acid and preferably, both the nitrite andthe acid in an aqueous solution are mixed together to which has beenadded effective amounts of additives such as surfactants, coloringagents, chelating agents and gelling agents, as otherwise describedherein. Metal nitrite salts are preferred for use in the presentinvention.

The term “nitrite ion” is used throughout the specification to describethe nitrite anion of a nitrite salt. In the present application, wherethe term “nitrite ion” is described in amounts in a given aqueouscomposition, it is the amount or concentration of the anion which isbeing referenced, not the amount of total salt concentration whichgenerally contains both a nitrite anion and a metal cation.

The term “acid” is used throughout the specification to describe proticacids, i.e., acids that release hydrogen ions in solution. Acids for usein the present invention include strong inorganic acids such ashydrochloric, sulfuric, and nitric acid; alkylsulfonic acid andbenzenesulfonic acid, among other organic sulfonic acids, which,depending upon the end-use of the composition, may be preferablyincluded as dilute acid; organic acids such as citric, fumaric,glycolic, lactic, malic, maleic, tartaric acid, salicylic, citric,propionic, acetic and mandelic, among others, includingethylenediaminetetraacetic acid (EDTA, as the free acid or themonosodium salt), among others; and inorganic acids such as sodium andpotassium bisulfate (NaHS0₄ and KHS0₄) and phosphoric acid, amongnumerous others. It is noted that numerous additional acids may also beused in the present invention. In its broadest aspect, compositionsaccording to the present invention may make use of virtually any acid,to the extent that it provides an initial pH, which when thenitrite-containing part and the acid-containing part are combinedproduce nitrous acid in amounts effective for the intended purpose. Oneof ordinary skill will be able to readily determine the type and amountof acid to be used for a particular application.

The term “effective amount” is used to describe that amount of acomposition, an individual component or a material which is included incompositions according to the present invention in order to produce anintended effect. For example, in the case of an effective amount of anacid, an effective amount is that amount which is included to produce asufficiently acidic medium to produce nitrous acid in combination with anitrite salt An effective amount of nitrite or a nitrite salt is thatamount which is effective to produce a desired concentration of nitrousacid after mixing with an appropriate and effective amount of an acid.In the case of a gelling agent, an effective amount of that component isthat amount which is effective to gel a final composition (i.e., producea viscous composition). One of ordinary skill will be able to readilydetermine effective amounts of components or compositions for use toprovide an intended effect.

The term “gelling agent” is used throughout the specification todescribe a compound or composition which is added to the presentcompositions in order to increase the viscosity of the composition.Gelling agents which are used in the present invention may be added tothe nitrite-containing part or the acid-containing part in amountseffective to gel the solution to which these compounds have been added.Gelling agents for use in the present invention include polysaccharidesproduced by microbial cultures such as xanthan, or extracted from legumeseeds, such as the galactomannans, including guar gum and locust bean(carob) gum. Other gelling agents include high molecular weightpolyoxyalkylene crosslinked acrylic polymers as well as the highlypreferred cellulosics such as hydroxymethyl cellulose, hydroxypropylcellulose, hydroxyethyl cellulose, methyl cellulose, methylpropylcellulose, among others, including high molecular weight polyethyleneglycols, polyacrylainide and polyacrylamide sulfonates, and crosslinkedpolyvinylpyrrolidones, among others. A gelling agent is used in aneffective amount in compositions to increase the viscosity of thecomposition. In a teat dip aspect of the invention, the amount ofgelling agent which is used is that amount which allows the compositionto cling to the teat of the cow or other mammal without significant lossof material from the exposed tissue. In preferred aspects of the presentinvention, the thickened composition remains on the teat upon drying ofthe composition thereon.

The term “storage stable” or “long-acting” refers to compositionsaccording to the present invention which exhibit significantantimicrobial activity (i.e., the composition can produce at least a 50%microbial kill within a period of no more than about 5 minutes andpreferably no more than about a minute or two) for a period of at leastabout 24 hours, preferably at least about 48 hours, preferably at leastabout 3 days (72 hours), preferably at least about 1 week (168 hours),and more preferably at least about three weeks (about 500 hours), atleast about two months, at least about three months or more. It is atleast one important aspect of the present invention to provide storagestable antimicrobial compositions which can be used as storage-stable orlong-acting compositions, in order to promote efficiencies and favorableeconomics of use.

The term “noncorrosive” or “substantially non-corrosive” are usedinterchangeably within the context of the present invention to describethe favorable characteristic of the invention as reducing orsubstantially avoiding corrosion of metal containers, which typicallyoccurs with the use of chlorous acid solutions on metal containers andother acid-sensitive equipment, especially milk containers and otherdairy equipment.

As described above, the present invention is directed to methods andprocesses for use of nitrous acid generating compositions to disinfectaninial teats and milking-associated equipment as well as for generaldisinfection purposes.

The composition comprises an aqueous solution containing a suitableamount of hydrogen ions derived from a protic acid and a suitable amountof a metal nitrite such as sodium nitrite. Compositions according to thepresent invention are preferably produced by adding a metal nitrite(either as a dry material or in solution) to an acidic aqueous solution.The concentration of hydrogen ion-generating species is such that theamount of nitrite ion in the form of nitrous acid is no more than about95% by weight of the total nitrite ion in the solution. Preferably, theamount of nitrite in the form of nitrous acid is no more than about 67%by weight of the total nitrite ion concentration in solution.

The percent by weight of nitrite and nitrous acid may be calculated fromthe ionization constant of nitrous acid and the amount of hydrogen ionin solution produced by partial ionization of the protic acid, orcalculated from the pH of a salt-induced acid solution. The hydrogen ionconcentration, [H⁺], in a solution of a protic acid, HA, of known molarconcentration and whose ionization constant is Ka, may be calculatedfrom the following relationship:${Ka} = \frac{\lbrack H^{+} \rbrack\lbrack A^{-} \rbrack}{\lbrack{HA}\rbrack}$

The above relationship may be applied to calculate the relative nitriteand nitrous acid concentrations, where the ionization constant fornitrous acid is 4.5×10⁻⁴. That is:${4.5 \times 10^{- 4}} = \frac{\lbrack H^{+} \rbrack\lbrack {{NO}_{2}}^{-} \rbrack}{\lbrack {HNO}_{2} \rbrack}$

where the hydrogen ion concentration, [H⁺], is the quantity readilydetermined by ionization of the known amount of the protic acid, HA.This calculation is well known to those skilled in this art.

For the nitrous acid/nitrite system, the following table illustratesrepresentative percentages of both species over a pH range whichprovides high to low amounts of nitrous acid, as derived from nitrite.TABLE 1 Percentage of Nitrite as Nitrous Acid at Varying pH Values pHNitrous Acid % Nitrite % 1.5 98.4 1.6 2.0 95.2 4.8 2.3 90.9 9.1 2.6 83.316.7 2.8 76.0 24.0 3.0 66.7 33.3 3.3 50.0 50.0 3.5 38.8 61.2 4.0 16.683.4 4.5 6.0 94.0 5.0 2.0 98.0

Aqueous solutions of nitrous acid are unstable, and decompose accordingto the following equation. Instability increases with increased absoluteand relative molar concentrations of the HONO, and with increasing heat:3HNO₂(aq)< - - - >H⁺(aq)+2NO(g)+NO₃ ⁻(aq)+H₂O(l)   Equation 1

The reaction is a combination of the two half-reactions, as follows:$\begin{matrix}{2\{ {{{HNO}_{2} + H^{+} + e^{-}} < \ldots > \quad{{NO} + {H_{2}O}}} \}} & {{Equation}\quad 2} \\\frac{{{HNO}_{2} + {H_{2}O}} < \ldots > {{NO}_{3^{-}} + {3H^{+}} + {2e^{-}}}}{\begin{matrix}{\Sigma = {{3{{HNO}_{2}({aq})}} < \ldots > {{H^{+}({aq})} +}}} \\{{2{{NO}(g)}} + {{{NO}_{3}}^{-}({aq})} + {H_{2}O\quad(1)}}\end{matrix}} & {{Equation}\quad 3}\end{matrix}$

In addition to the concentration-dependent degradation of nitrous acidas shown above, nitrous acid will also act as an oxidizing agent in thepresence of oxidizable materials, such as microorganisms, according tothe first half-cell reaction above (Equation 2), with a redox potentialε_(o)=1.00 volts. Accordingly nitrous acid systems are quite destructiveof all classes of microorganisms which are susceptible to oxidation,including bacteria, yeasts, molds and viruses. This destruction is wellknown for other non-specific oxidizing germicides such as bleach(hypochlorous acid), chlorous acid, chlorine dioxide, and iodine. Wehave discovered that acidified nitrite solutions, upon standing, willgenerally become either more acidic or less acidic in rough proportionto the pH of the solution, and thus the relative amount of nitrous acidwith respect to nitrite ion. At relatively high concentrations ofnitrous acid with respect to total nitrite (>˜75%), stored solutionswill generally become more acidic. At relatively lower concentrations ofnitrous acid, the stored solutions will generally become more alkaline(i.e. less acidic). In one set of experiments the break-point withrespect to greater or lesser acid formation occurred at ˜pH 3.7, wherethe total molar concentration of nitrite [ionic and acid-form] was0.045M/liter. The data were as follows: pH at T = 0 pH at T = 30 days2.94 2.30 3.12 2.50 3.35 3.25 3.54 3.15 3.75 3.92 3.90 4.35

The first solution, at pH 2.94, with a relative nitrous acid level ofabout 70% (see Table 1), increased in acidity to 2.30, a pH drop of 0.64units, whereas the last solution, at pH 3.90, and a relative nitrousacid level of about 20%, increased in pH by 0.45 units. The quantity ofacid required to reduce the pH in the first solution is, of course, muchgreater than for the last solution, in large measure because of thelogarithmic basis for the pH scale.

Although the direct reason for this difference is not fully understood,the increase or decrease of solution pH is believed to be related to thecorresponding contributions of half-ell Equations 2) and 3) above, theformer reducing the H⁺ present in solution (i.e. raising the pH) and thelatter contributing H⁺ to the medium, and thereby lowering the pH. Theremay be some involvement of the organic acidifier, which in thisexperiment was malic acid, in the overall reaction characteristic of theparticular combination of nitrite and acid concentrations in this set ofsolutions. However it is evident from this experiment that it isfeasible to adjust the concentrations of nitrite and acid in a preferredcomposition of this invention such that the solution is stabilized in pHover a prolonged period of time, and capable of being stored as apre-mixed one-part composition. This is a most-important finding, withrespect to the use of acidified nitrite solutions for teat careproducts, as pointed out earlier. This would be of significant benefitto dairy farmers, since a teat dip or spray with this type of stabilitywould obviate the need for daily, or even twice-daily mixing of ample,but not excess quantities sufficient for the particular set of animalsthat require pre- &/or post-milking dipping.

Nitric Oxide [NO], a paramagnetic species, loses an electron rathereasily, to form NO⁺, a reactive species. This reductive tendency is incontrast to the oxidative tendency of chlorine dioxide [ClO₂], anotherparamagnetic molecule which is a degradation product of chlorous acid.Therein lies a possible reason for the lower corrosion potential of theacidified nitrite system vs. that of acidified sodium chlorite. It isnot known, at this point, what aspect(s) of the acidified nitrite systemis/are the source of the antimicrobial activity which we haveestablished for this composition, although it appears reasonable thatthe NO and NO⁺ components play a significant role.

Without being limited by way of theory, it is believed that thestorage-stable compositions according to the present invention providelonger duration activity as a consequence of holding the pH of thecomposition within a set range, and holding the concentration of acidand nitrite salt within useful and effective ranges. There will be atension between initial HONO formation and antimicrobial activity andthe ability of the composition to maintain effectiveness beyond aninitial period of at least 24 hours, preferably at least about 72 hours,more preferably at least about 168 hours (one week) or at least about500 hours (about three weeks). In the acidified NO₂ ⁻/HONO system, i.e.NO₂ ⁻+H⁺< - - - >HONO, where only a fraction of the NO₂ ⁻ has beenconverted to the HONO germicidal source, when the latter has beendepleted or consumed in solution, additional HONO forms from theresidual NO₂ ⁻. The greater the degree of initial conversion, as afunction of the system's pH, the lower the reservoir of NO₂ ⁻ and thelower the absolute amount of HONO that can subsequently form. But thegreater the initial HONO, the greater the potential cidal activity thatis available for the system initially. And the greater the potential forthe HONO in the system to degrade, inasmuch as stability depends on HONOconcentration. Conversely, the lower the initial HONO the greater thereservoir of NO₂ ⁻, the greater the stability (i.e. prolonged germicidalactivity) but the less the cidal activity will be displaced. Thus, theuse of acid activating systems which provide a reservoir of [H⁺] ions,such as a-hydroxy acids, or phosphoric acid, which are not fully ionizedinitially, allows for additional [H⁺] ions to combine with the NO₂ ⁻ inthe reservoir over longer periods of time. Of course, for applicationswhere only initial activity is needed, even mineral acids can serve asthe proton source, by selecting their concentrations such that the pH ofthe system lies in about the 2.5 to 5.0 range.

In certain embodiments of the invention which are directed to a one-partstorage-stable composition, the nitrous acid generating compositioncomprises from about 0.03 to about 0.70, and preferably from about 0.05to about 0.50 percent by weight of metal nitrite, and an effectiveamount of an acid having a pKa of from about 2.1 to about 4.8 tomaintain the pH of the composition at less than about 4.5, morepreferably from about 2.5 to about 4.0. In certain aspects of theinvention, the amount of acid ranges from about 0.03% to about 3.0% byweight of the final composition, but an amount of acid outside of thisrange may be used, depending upon the amount of nitrite salt used, aswell as the desired antimicrobial activity and/or storage stability(length of time maintaining activity) desired. At metal nitrite levelshigher than about 0.7%, the concentration of nitrous acid formed uponadmixture of a protic acid, in the typical pH range specified, may be inexcess of that required for the formation of a metastable nitrous acidteat dip solution. These higher concentrations of nitrous acid couldpromote the formation of nitrous oxide, and nitric oxide therefrom,through the degradation of nitrous acid at too rapid a rate, viz.3HNO₂< - - - >H⁺+2NO+NO₃ ⁻+H₂O; then: 2NO+O₂(air)< - - - >2NO₂

Such solutions would not be preferred for use as teat dips or sprays,where extended lifetime (i.e., storage stability) of at least one day,are generally preferred.

Any protic acid, or acidic environment otherwise created, may be used inthe present invention so long as the nitrite ion concentration limitsdescribed above are met. Suitable protic acids include such inorganicacids as phosphoric acid, and such a-hydroxy organic acids as citric,malic, lactic, tartaric, glycolic, mandelic or other structurallysimilar acids as described in Formula 1 hereinabove and hereinbelow, forconvenience. The pKa of these organic acids may be generally from about2.8 to about 4.2, and preferably from about 3.0 to about 4.0. Alsosuitable are such other acids as salicylic acid and acetic acid.

In certain preferred embodiments of the invention, an acid is used ofthe formula:

where R¹ and R² may be the same or different and may be selected fromthe group consisting of hydrogen, methyl, —CH₂COOH, —CH₂COO⁻, —CH₂OH,—CHOHCOOH, —C₆H₅, and —CH₂C₆H₅. The pK_(a) of the organic acid ispreferably from about 2.8 to about 4.8.

The amount of acid used in these teat care compositions should besufficient to lower the pH of the composition to less than about 4.5,typically from about 2.5 to about 4, and preferably from about 2.5 toabout 3.5. Single acids are generally used, but combinations may be usedas well. The range of compositions of the teat dips is broad, of course,since useful acids range from the relatively weak, such as acetic acidwith a pKa of 4.76, to the moderately strong, such as tartaric acid witha first pK_(a) of 3.03 and phosphoric acid with a first pK_(a) of 2.15.Singly-used mineral acids, which at very low levels are capable ofgenerating disinfecting compositions, are of limited use in theinventive teat dips, since their limited H⁺ reservoir capacity formaintaining continuous optimum pH buffering predisposes to dips withlifetimes of insufficient duration.

While any metal nitrite is useful in the present composition, the alkaliand alkaline earth nitrites are preferred because they are readilysoluble, readily available and inexpensive. Sodium nitrite, potassiumnitrite and ammonium nitrite are preferred. Sodium nitrite isparticularly preferred.

A mixed acidified nitrite teat care composition will generally contain anumber of other components to facilitate the benefits of thedisinfecting composition. The teat dip will generally be an unthickened,slightly- or significantly-thickened colored aqueous solution, in whichwater represents a sufficient enough component that the normalequilibrium of the nitrite ion and nitrous acid may exist. The teat dipmay be colored (as with e.g. FD&C Yellow #5 and Yellow #6) or not, andmay contain other additives such as chelating agents (e.g. Na₂H₂EDTA),surfactants (e.g. alkyl aryl sulfonates such as Nacconol, and nonionicpolyoxyalkylene nonylphenols such as Triton N-101), preservatives (e.g.sodium benzoate), gelling agents or thickeners (e.g. cellulose ethers orxanthan), film-forming agents (e.g. polyvinylpyrollidone, among others),and opacifying agents or opacifiers (e.g. titanium dioxide or otheropacifying complexes such as those opacifying complexes formed by thereaction of cationic organic quaternary ammonium compounds (“quats”) andanionic organic sulfonates, among numerous others.

The nitrous-acid generating compound, i.e. the metal nitrite, isgenerally kept separate from the acid prior to use, in order to avoidpremature reaction of the ingredients. Thus, in a general aspect of thepresent invention, compositions prior to formation of nitrous acid arefound in a two part mixture. In general, once the two parts are mixed,there is an initial formation of nitrous acid followed by degradation ofthe nitrous acid, at a rate dependent on such factors as time,temperature, pH ands concentration of the nitrous acid. The latter willdepend upon both the absolute concentration of nitrite ion and theacidity of the system, which determines the degree to which the nitriteion is converted to nitrous acid, as demonstrated in Table 1hereinabove. At the upper range of pH values, of about 4.5, thesolutions may provide low levels of germicidal activity for severalmonths. At somewhat lower range of pH values, of about 3.5, the highinitial cidal capacity of the resulting solution may diminish with time,or even increase, as noted earlier. The molar concentration of nitritein the teat dip solution apparently plays a role in that change. Asnoted in an example described hereinbelow, where the total nitriteconcentration of one test solution [not a teat dip, per se] was 0.045M/liter, the initial solution at a pH of ˜3.7 actually increased incidal capacity. It demonstrated an E. coli kill actually greater at 20days after mixture, than the initial kill. And in a further example, itwill be noted that a barrier teat dip of the inventive composition,demonstrated an organism kill two weeks after preparation, equal to orbetter than the initial very high 8.5 log kill in 1 minute of contact.

The pre-mixes may also be combined by in situ application of theindividual parts. They may also be applied to the various substratesassociated with milking, such as teats and milking equipment, in amanner known to those skilled in this art. They may be dipped, sprayed,coated or applied in any other manner depending upon the substrate beingtreated. The term “substrate” as used in the instant specification isintended to cover any type of surface or carrier which could provide alocus for the accumulation of germs (bacteria, yeasts, molds, viruses,—i.e., all types of infectious agents).

Antimicrobial action may be enhanced or extended by inclusion of avariety of agents in either of the pre-mix acid or metal-nitritecompositions, or in the final mixture. These agents may include surfaceactive materials, chelating agents, effervescent compounds andthickeners. These materials must have a minimum tendency to react withthe nitrous acid system, or the acidic materials, and be compatible withthe other materials in the solutions. The surface active agents, or“surfactants” may be selected from the range of available classes, butnon-ionic and anionic surfactants are particularly effective. The amountof surfactant, on the final mix basis, is preferably in the range ofabout 0.001% to about 2%, more preferably no more than about 0.10%within this range, the level depending on the nature and effectivenessof the material in reducing the surface tension of the composition forthe desired application. When the surfactant is included in the teat dipto provide supplemental antimicrobial action, such as from alkyl arylsulfonates, the use level may be several orders of magnitude higher.

Preservatives may also be used in either or both of the pre-mixcompositions, to stabilize the solutions. On the basis of the totalcomposition the amount of preservative, from both pre-mix compositionsif so present, may generally be from about 0.01 to about 0.08, typicallyfrom about 0.01 to about 0.06, and preferably from about 0.02 to about0.04 percent by weight of the total composition. When the preservative,however, is included in the teat care composition to providesupplemental antimicrobial action, such as from sodium benzoate whichconverts to germicidal benzoic acid in an acid environment, the uselevel may be several orders of magnitude higher.

When these compositions are used as so-called “barrier” teat dips, theyare typically applied as thickened solutions to facilitate adherence tothe skin, and facilitate a greater laydown of germicide. Any thickeneror gelling agent which is non-toxic and non-reactive with the nitrousacid system may be used. Many carbohydrate polymers are possiblecandidates, although some such as the cellulose-based thickeners areless preferred because of their tendency to oxidatively cleave at theβ-D-glucose linkage. A preferred thickener is xanthan gum, which isminimally reactive in both the individual pre-mix composition and thefinal acidified nitrite mix. Other appropriate thickeners include thosebased on poly(oxyalkylenes) and poly(acrylamides) the latter includingthe sulfonic acid derivatives thereof, and mineral thickeners such asthe silica-based and clay gelling agents. Materials such as thepoly(acrylamides), and co-polymers thereof, can function as well as tocreate a skin-like covering on the teat, as the teat dip dries. Polymerssuch as poly(vinyl alcohol), which are non-thickening per se willsimilarly form pseudo-skins on the teat as the result of drying thereon.

The amount of thickener or gelling agent which may be used in thethickened, gel composition will vary, depending upon the thickeningproperties of the gelling agent, the intended application, the level andnature of the acid, the level of the metal nitrite, and other additivesemployed. Generally, the amount may be from about 0.5 to about 30,typically from about 1 to about 15, and preferably about 1 to about 12percent by weight of the total composition. Different thickeners may beused in each part of the pre-mix composition, and these levels refer tothe combined levels of gelling agent in the total composition.Film-forming agents may be used at about the same concentrations asthickeners or gelling agents in the present compositions.

The amount of metal nitrite in the nitrous-acid generating pre-mix isadjusted so that when the solution (including thickened liquid) is mixedwith the acidic component, the specified percentage of metal nitritewill be present in the resulting composition. For example, when twothickened pre-mixes are designed to be mixed in equal parts, which ispreferred, the amount of metal nitrite in one part may be generally fromabout 0.02% to about 2%, typically from about 0.04% to about 1%, andpreferably from about 0.06% to 0.6% by weight of that part. Similarly,the amount of acid in the counterpart pre-mix should be sufficient suchthat when that pre-mix is combined with the metal nitrite pre-mix, thepH of the resulting composition will be less than about 4.5, typicallyfrom about 2.5 to about 4, and preferably from about 2.5 to about 3.5.The wide diversity of possible acid sources is such that no particularweight specification for amounts of acid is feasible except on acase-by-case basis, although the acid is used in the present inventionin effective amounts.

The two teat-dip pre-mix liquids may be combined just prior toapplication, or up to at least several weeks before use, or they may besimultaneously mixed and applied in situ. The teat care compositions maybe dipped, sprayed, or may be coated onto teats by techniques known tothose skilled in dairy practices, or applied in any other mannerdepending upon the needs of the dairy practitioner or farmer.

In certain preferred embodiments of the present invention a disinfectantcomposition comprises a single-phase liquid or gel comprising nitrousacid and an a-hydroxy acid, wherein the pH of the composition eitherremains relatively constant at an initial value of around 3.7 or lower,or decreases from said initial value of around 3.75 or lower at the timeof formulation to a value as low as around 2.5 over a period of at leastabout two days, preferably about two days to five days; the molarpercentage of nitrite ion in the composition in the form of nitrous acidis greater than about 35% but less than about 95% of the total nitriteions present in the composition; and the composition exhibits cidalactivity against microorganisms for a period of at least three weeks(preferably at least about two months or at least three months) afterformulation.

The present invention is illustrated by the following Examples. Examples1 through 5, and Example 9 illustrate the basic germicidal capabilitiesof the acidified nitrite systems, and Examples 6,7 and 8 demonstrate thefunctionality of acidified nitrite teat care systems. All parts andpercentages in the Examples, as well as the specifications and claims,are by weight, unless otherwise specified. The following examples, whichare non-limiting, further describe preferred embodiments within thescope of the present invention. Many variations of these Examples arepossible without departing from the spirit of the invention.

EXAMPLE 1

This example illustrates the ability of six acidified nitrite solutionsto destroy high levels of the Gram-positive organism Staphylococcusaureus (ATCC 29213), and to a degree consistent with the relativepercentage of nitrous acid with respect to total nitrite in thesolution. The mixed nitrite/acid solutions, their resulting pH values,and the relative percentages of nitrous acid in the solutions were asshown below. To prepare these solutions, equal parts of a 0.625% NaNO₂solution and increasing concentrations of malic acid solution werecombined as follows: Malic Total Nitrite Sol'n No. NaNO₂ Premix AcidPremix Mix pH as Nitrous Acid 1 0.625%  2.25% 2.94 70% 2 0.625% 1.225%3.12 60% 3 0.625% 0.812% 3.35 47% 4 0.625% 0.419% 3.54 37% 5 0.625%0.263% 3.75 28% 6 0.625% 0.156% 3.90 21%

Procedure: A heavy suspension of the S. aureus was prepared in saline,and 1 part of the suspension was separately combined with 10 parts ofeach of the above solutions, which had been prepared five minutes beforethe testing. After five minutes of contact, the mixtures were added tonine volumes of Dey/Engley broth to neutralize the activity and acidity.A 10-fold dilution in saline was made of this mixture. 2 mls of thesample diluted in D/E broth were added to each of five petri plates. 1ml of the sample diluted in D/E broth was added to each of two petriplates, and 1 ml of the 1/10 dilution of the sample diluted in D/E brothwas added to each of two petri plates. Approximately 10 mls of semisolidTrypticase Soy Agar were added to each petri plate, swirled and allowedto harden. The plates were incubated at 350-37° C. for 48 hours, and theresulting colonies were enumerated.

The number of microorganisms in the original suspension was determinedby making ten-fold dilutions from 10⁻¹ to 10⁻⁸. Then 1.0 ml portions ofthe 10⁻⁷ suspension were added to each of two sterile petri plates. 1.0ml of the 10⁻⁸ suspension was added to each of two sterile petri plates,and 0.1 ml of the 10⁻⁸ suspension was added to each of two sterile petriplates. Approximately 10 mls of semisolid agar were added to each petriplate, swirled and allowed to harden. The plates were incubated at35°-37° C. for 48 hours, and the resulting colonies were enumerated.Results: S. aureus Cidal Data* Sol'n No. Recovered cfu Log Recovery LogKill 1 5.4 × 10¹ 1.7 9.1 2 7.0 × 10³ 3.8 7.0 3 4.5 × 10³ 3.6 7.2 4 5.6 ×10⁴ 4.7 6.1 5 6.6 × 10⁵ 5.8 5.0 6  >1 × 10⁶ >6.0 <4.8*Inoculum suspension contained 10.8 logs of organisms.

It is obvious that a)—there was significant destruction of the highinoculum of S. aureus in the 5-minute contact period, and b)—the degreeof destruction closely parallels the degree of conversion of the nitriteion to nitrous acid. A 9.1 log kill (>1 billion-fold) was achieved witha solution in which 70% of the nitrite existed in its acidified form ofnitrous acid, whereas only 5.0 logs (100,000-fold) were destroyed by thesolution with nitrous acid representing 28% of the total nitrite. Evenless was destroyed in the 21% nitrous acid (relative) solution.

EXAMPLE 2

This example illustrates the ability of six acidified nitrite solutionsto destroy high levels of the Gram-negative organism Escherichia coli(ATCC 25922). The procedure described in Example 1 was applied in thisstudy as well, using aliquots of the same solutions described in theTable.

The results were as follows: Results: E. coli Cidal Data* Sol'n No.Recovered cfu Log Recovery Log Kill 1 2.7 × 10² 2.4 7.7 2 6.6 × 10⁴ 4.85.3 3 9.0 × 10⁰ 1.0 9.1 4 1.4 × 10¹ 1.1 9.0 5 9.9 × 10² 3.0 7.1 6 3.1 ×10³ 3.5 6.6*Inoculum suspension contained 10.1 logs of organisms.

In the case of this Gram-negative organism, the destruction of theinoculum was high in all solutions, apparently independent of pH andthus the relative amount of total nitrite existing as nitrous acid inthis series of solutions. It is not known, at this point, whether thisdifference with respect to the observations in Example 1 ischaracteristic of the kill mechanism of acidified nitrite solutions withrespect to Gram-positive and Gram-negative organisms, or whether itrelates to these particular organisms

EXAMPLE 3

This example illustrates the ability of six acidified nitrite solutionsto destroy high levels of the Gram-negative organism Escherichia coli(ATCC 25922), following 20 days of storage of the mixed solutions atambient temperatures prior to the testing. The procedure described inExample 1 was applied in this study as well, using aliquots of the samesolutions that were evaluated in Examples 1 and 2. The results were asfollows:

Results:

The data are presented in the following Table, in which the killsmeasured on the 20-day old solutions are compared with data obtained onthe T=0 mixtures (in brackets). E. coli Cidal Data on 20-day agedmixtures* Sol'n No. Recovered cfu Log Recovery Log Kill** 1 6.0 × 10¹1.8 9.2 [7.7] 2 1.5 × 10² 2.2 8.8 [5.3] 3 6.0 × 10⁰ 0.8 10.2 [9.1]  4 >1 × 10⁵ >5.0 <6.0 [9.0]  5 3.2 × 10⁴ 3.5 7.5 [7.1] 6  >1 × 10⁶ >6.0<5.0 [6.6] *Inoculum suspension contained 11.0 logs of organisms.**Bracketed data are log kills at T = 0 with the same solutions

About three weeks after preparation, the mixed solutions have retained asignificant cidal capacity, as compared with their abilities at T=0. Infact the pH's of these aged solutions, as cf. their original values,sheds some light on the greater cidal capacity of the first fewsolutions tested, viz. pH at T = 0 pH at T = 30 days 2.94 2.30 3.12 2.503.35 3.25 3.54 3.15 3.75 3.92 3.90 4.35

The highest activity, in both fresh and aged solution, appears to occurin the solutions where the pH levels dropped, leading to higher levelsof nitrous acid. In these solutions, the nitrous acid and nitrite existin a ratio of ca. 1:1 and higher. This leads to the speculation that thestability (as well as the activity) of these solutions is related to thepresence of a complex ion, such as [HN₂O₄]⁻, analogous to the [Cl₂O₄]⁻found to exist in ClO₂/ClO₂ ⁻ systems, where the complex [Cl₂O₄]⁻ isconjectured to be an active cidal species, of a higher oxidationpotential than ClO₂ alone.

EXAMPLE 4

This example illustrates the ability of six acidified nitrite solutionsto destroy high levels of the yeast Candida albicans (ATCC 10231), andto a degree consistent with the relative percentage of nitrous acid withrespect to total nitrite in the solution. The mixed nitrite/acidsolutions, their resulting pH values, and the relative percentages ofnitrous acid in the solutions were similar to those shown in Example 1.

Procedure: A heavy suspension of the C. albicans was prepared in saline,and 1 part of the suspension was separately combined with 10 parts ofeach of the above solutions, which had been prepared five minutes beforethe testing. After five minutes of contact, the mixtures were added tonine volumes of Dey/Engley broth to neutralize the activity and acidity.A 10-fold dilution in saline was made of this mixture. 2 mls of thesample diluted in D/E broth were added to each of five petri plates. 1ml of the sample diluted in D/E broth was added to each of two petriplates, and 1 ml of the 1/10 dilution of the sample diluted in D/E brothwas added to each of two petri plates. Approximately 10 mls of semisolidSabouraud Dextrose Agar were added to each petri plate, swirled andallowed to harden. The plates were incubated at 20°-25° C. for 72 hours,and the resulting colonies were enumerated.

The number of microorganisms in the original suspension was determinedby making ten-fold dilutions from 10⁻¹ to 10⁻⁸. Then 1.0 ml portions ofthe 10⁻⁷ suspension were added to each of two sterile petri plates. 1.0ml of the 10⁻⁸ suspension was added to each of two sterile petri plates,and 0.1 ml of the 10⁻⁸ suspension was added to each of two sterile petriplates. Approximately 10 mls of semisolid agar were added to each petriplate, swirled and allowed to harden. The plates were incubated at20°-25° C. for 72 hours, and the resulting colonies were enumerated. C.albicans Cidal Data* Sol'n No. Recovered cfu Log Recovery Log Kill %HONO** 1 0 0 >7.86 70 2 4 0.6 7.26 60 3 2.4 × 10¹ 1.38 6.48 47 4 2.1 ×10⁴ 4.32 3.54 37 5  >1 × 10⁶ >6 <˜1 28 6  >1 × 10⁶ >6 <˜1 21*Inoculum suspension contained 7.86 logs of organisms.**% of total nitrite ion present as nitrous acid

The destruction of the C. albicans yeast is quite significant,particularly for the solutions below about pH 3.5, where the nitrousacid is present in a ratio of about 1:1 with respect to ionic nitrite(i.e., above about 50% of total nitrite as HONO). Thereafter the falloff in kill is rather dramatic, at higher pHs. For this organism, as forthe S. aureus of Example 1, this suggests that a 1:1 adduct of nitrousacid and nitrite may be providing particularly effective cidal capacityin this system.

EXAMPLE 5

This example illustrates the ability of six acidified nitrite solutionsto destroy high levels of the mold Aspergillus niger (ATCC 6275). Themixed nitrite/ acid solutions, their resulting pH values, and therelative percentages of nitrous acid in the solutions were similar tothose shown in Example 1, and the procedure followed paralleled thatprovided in Example 4. A. niger Cidal Data* Sol'n No. Recovered cfu LogRecovery Log Kill 1 18 1.26 7.14 2 83 1.92 6.48 3 30 1.48 6.92 4 37 1.576.83 5 0 0 >8.40 6 0 0 >8.40*Inoculum suspension contained 8.40 logs of organisms.

EXAMPLE 6

This example illustrates the high level and duration of efficacy of anacidified nitrite teat dip composition against the Environmentalorganism E. coli (ATCC 25922). An in vitro microbiological evaluationwas run on the composition at three times; when freshly mixed as well as1 day and 2 days after preparation. The two components of the teat dipwere as follows: Nitrite Base: Sodium nitrite 0.625%  Sodiumdodecylbenzene sulfonate 0.20% FD&C Yellow #5 0.20% Water q.s. AcidActivator: Lactic acid (88%)* 3.23% Glycerin 10.0% Natrosol 250MR 0.50%Sodium benzoate 0.04% Benzalkonium chloride (17%) 1.26% Water q.s.*HCl was added so that a 1:1 mix of both parts had a pH of 2.95.

Procedure: The initial inoculum at each test period was >10⁸, as will beseen in the test data. The microorganism was plated on Trypticase SoyAgar and incubated at 35°-37° C. for 24 hours. A heavy suspension wasprepared in sterile saline. Equal quantities (by weight) of the teat dipcomponents were mixed together, and allowed to stand for about 10minutes. Then nine volumes of this sample was challenged with one volumeof the organism suspension for 15 seconds. Then 2.0 ml of the mixturewere added to 18 ml of D/E broth. A further 1/10 dilution of the D/Ebroth in saline was prepared. Five 2.0 ml samples of the D/E broth wereadded to petri plates. Duplicate 1.0 ml samples were added to petriplates, and duplicate 1.0 ml samples of the 1/10 dilution were added topetri plates. Approximately 10 ml of liquid Trypticase Soy Agar wereadded to each petri plate and allowed to solidify. Plates were incubatedat 35°-37° C. for 24-48 hours, and colony forming units were counted.Thereafter the mixed sample was incubated in a foil-covered sterilecontainer at room temperature, until use. After the first sample (Day 0)sample was tested, samples were removed for testing 1 and 2 days aftermixing (Day 1 and 2, resp.) and were tested as above. At each test pointa control study was run, in which a sample of saline was challenged,instead of the test sample. Results: Challenge Inoculum Organisms (Log#cfu/ml Recovered (Log Log Test Sample Product) #cfu/ml ProductReduction Day 0 Teat Dip 7.8 × 10⁸ (8.89) 0 >8 89 Control (Saline) 7.8 ×10⁸ (8.89) 3.8 × 10⁸ (8.89) — Day 1 Teat Dip 5.3 × 10⁸ (8.72) 1.7 × 10¹(1.23)  7.47 Control (Saline) 5.3 × 10⁸ (8.72) 5.1 × 10⁸ (8.70) — Day 2Teat Dip 3.4 × 10⁸ (8.53) 0 >8.56 Control (Saline) 3.4 × 10⁸ (8.53) 3.6× 10⁸ (8.56) —

These results clearly demonstrate that the acidified nitrite teat dipwas capable of destroying upwards of 100 million E. coli organismswithin 15 seconds of contact, up through two days following mixture. The17 remaining organisms, of the 530 million challenge at Day 1, areconsidered artifactual, in as much as the 2-day aged sample destroyedall of the challenge. It is evident from these data that acidifiednitrite antimicrobials can exert continued cidal activity againstmastitis-causing microorganisms long after their initial preparation.

EXAMPLE 7

This example illustrates the prolonged high-level efficacy of athickened version of the above acidified nitrite teat dip compositionagainst the Environmental organism E. coli (ATCC 25922). This type ofteat dip is generally termed a “barrier” dip, because it deposits aprotective film on the teat during and after drying, so as to protectthe teat during the intermilking period. The composition provided inExample 6 was modified by the addition of two components to the nitritebase, specifically 0.50% xanthan gum and 2.24% of Fixomer A-30, a 70/30copolymer of methacrylic acid and poly(acrylamidomethyl propane sulfonicacid). In this study, in vitro microbiological evaluations were run onthe composition at five times; when freshly mixed as well as 1, 2, 6 and14 days after preparation. The procedure was the same as in Example 6,except that a 1-minute contact was used for the studies, based on theextended contact of a barrier dip, which is applied post-milking, andintended to last on the teat for up to ˜12 hours until the next milking.Results: Challenge Inoculum (Log Organisms Recovered Log Test Sample#cfu/ml Product) (Log #cfu/ml Product) Reduction Day 0 Teat Dip 2.2 ×10⁸ (8.34) 0 >8.40 Control (Saline) 2.2 × 10⁸ (8.34) 2.5 × 10⁸ (8.40) —Day 1 Teat Dip 4.0 × 10⁸ (8.60) 1.7 × 10¹ (1.23)  7.20 Control (Saline)4.0 × 10⁸ (8.60) 2.7 × 10⁸ (8.43) — Day 2 Teat Dip 3.4 × 10⁸ (8.53)0 >8.38 Control (Saline) 3.4 × 10⁸ (8.53) 2.4 × 10⁸ (8.38) — Day 6 TeatDip 3.2 × 10⁸ (8.51) 0 >8.61 Control (Saline) 3.2 × 10⁸ (8.51) 4.1 × 10⁸(8.61) — Day 14 Teat Dip 7.8 × 10⁸ (8.89) 0 >8.58 Control (Saline) 7.8 ×10⁸ (8.89) 3.8 × 10⁸ (8.58) —

These results clearly demonstrate that the acidified nitrite barrierteat dip was capable of destroying 220-780 million E. coli organismswithin 60 seconds of contact, up through two weeks following mixture. Asdemonstrated in Example 6, and further evident from these data,acidified nitrite teat dips can exert continued and very high cidalactivity against mastitis-causing microorganisms long after the teatdip's initial preparation.

EXAMPLE 8

This example demonstrates the antimicrobial activity of the teat dipcomposition described in Example 6 against the Contagious microorganismStaph. aureus [ATCC 29213], using a teat model system. In this test,four wooden birch dowels are used, for both test and control samples, tosimulate animal teats. The microorganism suspension was prepared byinoculating 50 ml of Trypticase Soy Broth (TSB), and incubating at35°-37° C. for 18-24 hours. The wooden dowels, (ca. 0.6 inch diameter×2inch length) were fitted with screw hooks attached at one end atapproximately a 45° angle, to facilitate dripping of excess teat dip.The dowels were marked at a point one inch from the end opposite thehook, and then covered with the finger of a latex glove. Each dowel wasthen suspended from a wire.

At the beginning of each experiment, each dowel was sprayed with 70%isopropyl alcohol and allowed to dry for ca. 10 minutes. Following this,each dowel was dipped into the microorganism suspension, up to the oneinch mark. The dowels were again allowed to dry for approximately 10minutes. 15 ml of each of the teat dip components were mixed forapproximately 15 seconds, and allowed to stand for about 5 minutes. Thedowels were then dipped into the teat dip, past the one-inch mark, toensure that all of the dried microorganism had been covered. This wasreplicated on the remaining four simulated teats. After a one-minutecontact, the dowels were submerged past the one-inch mark in 18 ml ofD/E broth. A 1/10 dilution of the D/E broth was prepared in saline.

Duplicate 1.0 ml samples of the D/E broth were placed into each of twopetri plates. Duplicate 1.0 ml samples of the 1/10 dilution of the D/Ebroth were placed into each of the two petri plates, and duplicate 0.1ml samples of the 1/10 dilution of the D/E broth were placed into eachof two petri plates. Approximately 10 ml of liquid Trypticase Soy Agarwas added to each petri plate and allowed to solidify. Plates wereincubated at 35°-37° C., for 24-48 hours, and colony forming units werecounted. Log reductions of the test dip challenges were calculated withrespect to the organisms present in the Control saline. Although theinitial S. aureus challenge contained 7.2×10⁸ organisms per ml, thesaline itself physically removes several logs worth of organisms fromthe teat model, so the reference quantity is considerably smaller (ascan be seen from the following data tabulation). Geometric Reduction vs.Sample Teat 1 Teat 2 Teat 3 Teat 4 Average Control Test 0 0 0 0 0 5.4logs Control 2.8 × 10⁵ 2.6 × 10⁵ 2.2 × 10⁵ 2.5 × 10⁵ 2.8 × 10⁵ (5.4logs) —

These data confirm that the teat dip comprised of an acidified nitritesolution, is an effective cidal agent against a microorganism stronglyassociated with Contagious mastitis in dairy cows.

EXAMPLE 9

This example illustrates the ability of one of the six nitrous acidsolutions tested in Examples 1 through 5, specifically Solution No. 2,to be as microbiocidally effective after over two (2) years of storageat ambient temperatures, as it was in both Example 2 (the day ofpreparation) and Example 3 (after 20 days of ambient storage). InExample 2 the nitrous acid, formulated with equal parts of 0.625% NaNO₂and 1.225% Malic Acid, was shown to destroy 5.3 logs of theGram-negative organism Esciherichia coli (ATCC 25922) after 5 minutes ofcontact. In Example 3, after 20 days of storage, the aged solutiondestroyed 8.8 logs of that organism.

After over 26 months of ambient storage (specifically 735 days), analiquot of that nitrous acid solution was tested for its 5-minute kill,with the following results: Test Organism: E. coli ATCC 25922 InitialSuspension: 1.4 × 10⁹ Challenge Recovered Inoculum (Log (Log Log TestSample cfu/ml) cfu/ml) Reduction Sample mixed on Sep. 28, 2001 1.4 × 10⁸0 >8.15 and stored at room temp. (8.15 logs) Control (Saline) 1.4 × 10⁸1.1 × 10⁸ —

The procedure for E. coli was the same as described in the earlierExamples as follows:

The microorganism was plated on Trypticase Soy Agar and incubated at35-37° C., for 18-24 hours. A heavy suspension was prepared in sterilesaline. The challenge sample, which had been mixed on Sep. 28, 2001, hadbeen stored in a capped glass test tube at room temperature untiltesting. Nine volumes of the sample (1.8 ml) were challenged with onevolume (0.2 ml) of the organism for 5 minutes. Following this 2.0 ml ofthis mixture was added to 18 ml of D/E broth. A further 1/10 dilution ofthe D/E broth in saline was prepared. Five 2.0 ml samples of the D/Ebroth were added to petri plates. Duplicate 1.0 ml samples were added topetri plates, and duplicate 1.0 ml samples of the 1/10 dilution wereadded to petri plates. Approximately 10 ml of liquid Trypticase Soy Agarwas added to each petri plate and allowed to solidify. Plates wereincubated at 35-37° C., for 24 hours, and colony forming units werecounted. A control study was run, in which a sample of saline waschallenged, instead of the test sample.

This Example clearly demonstrates that this nitrous acid solution, at apH below about 3.4 (as deducted from the aging data in Example 3 and thepH information provided in Example 1), is capable of providing a highlevel of antimicrobial activity, for at least several years after itsformation, when stored under ambient conditions.

It is clear that the present invention is well adapted to carry out theobjects, and achieve the ends and advantages mentioned at the outset.While currently preferred embodiments of the invention have beendescribed for purposes of this disclosure, numerous modifications may bemade which will readily suggest themselves to those skilled in the art,and which are encompassed within the spirit of he invention disclosed,and as defined in the appended claims. References Cited US PatentDocuments Number Issue Date Inventor 4084747 April, 1978 Alliger 4330531May, 1982 Alliger 4891216 January, 1990 Kross et al. 4956184 June, 1988Kross 4986990 January, 1991 Davidson etal. 5100652 May, 1992 Kross etal. 5185161 February, 1993 Davidson et al. 5384134 January, 1995 Kross,et al. 5389390 February 1995 Kross 5597561 January, 1997 Kross 5628959May, 1997 Kross 5651977 July, 1997 Kross 5772985 June, 1998 Kemp, et al.5820822 October, 1998 Kross RE36,064 January, 1999 Davidson et al.6063425 May, 2000 Kross 6096350 August, 2000 Kemp, et al. 6099881August, 2000 Hanson 6123966 September, 2000 Kross Other Patent DocumentsPCT Application WO95/22335 Feb. 17, 1995 Nigel Benjamin et al.(Acidified Nitrite as an Antimicrobial Agent) PCT Application WO02/17881 Aug. 30, 2001 Tucker et al. (Transdermal PharmaceuticalDelivery System) US Patent Appl. Pub. US 2002/0136750 A1 Benjamin et al.Sep. 26, 2002 (Acidified Nitrite as an Antimicrobial Agent) US PatentAppl. Pub. US 2002/0155174 A1 Benjamin et al. Oct. 24, 2002 (AcidifiedNitrite as an Antimicrobial Agent)

OTHER REFERENCES

-   Friedman, H. L. “On tile Ultraviolet Absorption Spectra of    Uninegative Ions”, J. Chem. Physics, (1953), Vol. 21, No. 1, p. 319    et seq.-   Masschelein, W J; (1979) Chlorine Dioxide; Chemistry and    Environmental Impact of Oxychlorine Compounds. Ann Arbor Science,    Mich.

1. A process for disinfecting mammalian teat skin comprising contactingsaid substrate with an aqueous teat dip composition consistingessentially of water and an effective amount of a protic acid, or amaterial inducing an acidic environment therein, and an effective amountof a water soluble metal nitrite to produce nitrous acid from said acidand said metal nitrite, said composition containing an amount of nitrousacid which is no more than about 95% by weight of the total amount ofnitrite ion and nitrite as nitrous acid in said composition.
 2. Theprocess according to claim 1 wherein the metal nitrite is present at alevel of from about 0.03% to about 0.7% by weight based on the totalweight of the composition.
 3. The process according to claim 1 whereinthe metal nitrite is sodium nitrite.
 4. The process according to claim 1wherein the protic acid is an organic acid.
 5. The process of claim 4wherein the organic acid has a pK_(a) value in the range of about 2.8 toabout 4.8.
 6. The process of claim 4 wherein the organic acid has apK_(a) value in the range of about 2.8 to about 4.2.
 7. The process ofclaim 4 wherein the organic acid has a pK_(a) value in the range ofabout 3.0 to about 4.0.
 8. The process according to claim 4 wherein saidacid is an α-hydroxy acid of the general formula:

wherein R¹ and R² may be the same or different and may be selected fromthe group consisting of hydrogen, methyl, —CH₂ COOH, —CH₂ COO⁻, —CH₂ OH,—CHOHCOOH, —C₆H₅ , and —CH₂C₆H₅.
 9. The process of claim 5 wherein theorganic acid ranges from about 0.03% to about 3% by weight of the totalcomposition.
 10. The process according to claim 1 wherein said aqueouscomposition has a pH of less than about 4.5.
 11. The process accordingto claim 1 wherein said aqueous composition has a pH ranging from about2.5 to about 4.0.
 12. The process according to claim 1 wherein saidaqueous composition has a pH ranging from about 2.5 to about 3.5. 13.The process according to claim 1 wherein the protic acid is an inorganicacid.
 14. The process of claim 13 wherein the inorganic acid is selectedfrom the group consisting of nitric acid, hydrochloric acid, sulfuricacid, sodium hydrogen sulfate and phosphoric acid.
 15. The process ofclaim 1 wherein the teat dip composition is a thickened compositioncomprising a gelling or film-forming agent.
 16. The process according toclaim 15 wherein said thickened composition remains on the teat upondrying of the composition thereon.
 17. The process according to claim 15wherein said gelling or film-forming agent comprises about 0.5 percentto about 30 per cent by weight of said composition, typically from about1 percent to about 15 percent, and preferably at about 1 percent toabout 12 percent.
 18. The process of claim 1 wherein the aqueous teatdip composition retains significant microbiocidal activity for a periodof at least about 24 hours after preparation.
 19. The process of claim 1wherein the aqueous teat dip composition retains significantmicrobiocidal activity for a period of at least about three weeks afterpreparation.
 20. The process of claim 1 wherein the aqueous teat dipcomposition retains significant microbiocidal activity for a period ofat least about two weeks after preparation.
 21. The process according toclaim 1 wherein the mammalian skin is that of a cow.
 22. A long-actingantimicrobial composition comprising a single-phase liquid or gelcomprising an antimicrobially effective amount of nitrous acid and aprotic acid wherein said composition comprises about 0.03% to about0.70% by weight of a metal nitrite and said protic acid is included insaid composition in an amount effective to produce a pH of less thanabout 4.5 in said composition and wherein said antimicrobial activity ismaintained in said composition for a period of at least about 48 hours.23. The composition according to claim 22 wherein said acid is anα-hydroxy acid having a pKa value ranging from about 2.1 to about 4.8,said pH of said composition ranging from about 2.5 to about 4.0.
 24. Thecomposition according to claim 22 wherein said protic acid has thechemical structure:

wherein R¹ and R² may be the same or different and may be selected fromthe group consisting of hydrogen, methyl, —CH₂COOH, —CH₂COO⁻, —CH₂OH,—CHOHCOOH, —C₆H₅, and —CH₂C₆H₅.
 25. A composition according to claim 22,wherein the nitrous acid is generated by a sodium or potassium nitrite.26. A composition according to claim 22, wherein the composition is aliquid teat dip.
 27. A composition according to claim 22, wherein thecomposition is a gel.
 28. A composition comprising a single-phase liquidor gel comprising nitrous acid and an α-hydroxy acid, wherein: (a) thepH of the composition either remains relatively constant at an initialvalue of around 3.7 or lower, or decreases from said initial value ofaround 3.75 or lower at the time of formulation to a value as low asaround 2.5 over a period of at least about two days, preferably abouttwo days to five days; (b) the molar percentage of nitrite ion in thecomposition in the form of nitrous acid is greater than about 35% butless than about 95% of the total nitrite ions present in thecomposition; and (c) the composition exhibits cidal activity againstmicroorganisms for a period of at least three weeks after formulation.29. A composition of claim 28, wherein the composition comprises acompound comprising an amount of phosphoric acid with a pKa of about2.15 that is sufficient to lower the pH of the composition to less thanabout 3.75.
 30. A composition according to claim 28, wherein thea-hydroxy acid is a compound of the formula (I):

wherein R¹ and R² may be the same or different and may be selected fromthe group consisting of hydrogen, methyl, —CH₂COOH, —CH₂COO⁻, —CH₂OH,—CHOHCOOH, —C₆H₅, and —CH₂C₆H₅.
 31. A composition according to claim 28,wherein the composition further comprises one or more of the followingcomponents: a surface active agent, a chelating agent, an effervescentcompound, a preservative, a coloring agent, an opacifier and athickener.
 32. A composition according to claim 28, wherein the cidalactivity of the composition over a period of about twenty-four months ormore after formulation is comparable to the activity that itdemonstrated initially.
 33. A composition according to claim 28, whereinthe cidal activity of the composition over a period of about fiveminutes or more after formulation is equivalent to the activitynecessary to achieve an approximately eight log decrease in a sample ofE. coli.
 34. A composition according to claim 28, wherein thecomposition is used in conjunction with an application medium.
 35. Acomposition according to claim 28, wherein the nitrous acid is generatedby a metal nitrite.
 36. A composition according to claim 28, wherein thecomposition may be used as a liquid teat dip.
 37. A compositionaccording to claim 28, wherein the composition is a gel.
 38. A methodcomprising disinfecting a substrate by application thereto of acomposition according to claim
 28. 39. A method comprising disinfectingmammalian tissue by application to said tissue of a compositionaccording to claim
 28. 40. The method according to claim 38 wherein saidsubstrate is a metal surface.
 41. A method according to claim 38 whereinsaid composition comprises an amount of nitrite in the form of nitrousacid that is no more than about 85% by weight of the total nitrite ionsin the composition.
 42. The method according to claim 38, wherein thecomposition is a disinfecting gel comprising a thickener.
 43. The methodaccording to claim 38, wherein the composition is an oral rinse.
 44. Themethod according to claim 38 wherein said application of saidcomposition occurs over a period of at least about several months. 45.The method according to claim 38 wherein the substrate is mammaliantissue.
 46. The method according to claim 38 wherein the composition issprayed onto the substrate. 47-51. (canceled)